The goal of this Project is to develop recombinant antibody fragments capable of activating Toll-like receptors (TLRs) on the surface of dendritic cells (DC). These antibody fragments will be further developed into vaccine adjuvants by conjugation to appropriate target immunogens. We hypothesize that stimulation of specific DC TLRs by antibody-immunogen conjugates will deliver the immunogen in a manner that supports the induction of a protective immune response. Individuals vaccinated in this manner should then be protected from the infectious agents or toxins from which the immunogens were derived. This Project will develop activating antibody fragments for each of the ten known TLRs. Thus, for each TLR, we will 1) generate a mudne TLR-specific single chain antibody fragment (scFv) phage display library; 2) screen this library over appropriate human and murine TLR protein constructs; 3) determine the various epitope classes represented by the recovered scFvs; 4) develop bivalent scFv constructs, termed "diabodies" capable of activating DC; 5) optimize diabody affinity and constant region properties; 6) prepare diabody-immunogen conjugates for use in the other Projects; and 7) develop human diabody constructs with comparable DC activating properties. Work on each of the TLRs will proceed in series over the entire project period, so that insights and breakthroughs obtained from work on the first TLRs will expedite work on the later TLRs. This Project will result in a panel of recombinant antibody constructs capable of selective activation of discrete TLR classes and delivery of a specific immunogen to DC and other cell types expressing those TLRs. This adjuvant technology is fully modular and will be extensible to a wide range of infectious agents and toxins.